title: Single-cell analysis of thymocyte differentiation: identification of transcription factor interactions and a major stochastic component in αβ-lineage commitment.
abstract: T cell commitment and αβ/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αβ/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αβ/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αβ-committed cells do not express the pre-TCR and revealed a major stochastic component in αβ-lineage specification.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 1020, and Université Paris Descartes, Unité Mixte de Recherche, Paris, France.
authors.forename: Amine
authors.initials: A
authors.lastname: Boudil
authors.forename: Lamia
authors.initials: L
authors.lastname: Skhiri
authors.forename: Serge
authors.initials: S
authors.lastname: Candéias
authors.forename: Valérie
authors.initials: V
authors.lastname: Pasqualetto
authors.forename: Agnès
authors.initials: A
authors.lastname: Legrand
authors.forename: Marie
authors.initials: M
authors.lastname: Bedora-Faure
authors.forename: Laetitia
authors.initials: L
authors.lastname: Gautreau-Rolland
authors.forename: Benedita
authors.initials: B
authors.lastname: Rocha
authors.forename: Sophie
authors.initials: S
authors.lastname: Ezine
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D020869:Gene Expression Profiling; D051379:Mice; D059010:Single-Cell Analysis; D013269:Stochastic Processes; D060168:Thymocytes / Q000166:cytology* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors